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Biomedical subjects

R Yanagimachi

Publications and source records attributed to R Yanagimachi.

At least 145 records · Page 8Linked to original sources
PubMed1986

Development of a cortical granule-free area of cortex and the perivitelline space in the hamster oocyte during maturation and following ovulation.

The distribution and behavior of cortical granules (CGs) in hamster oocytes at various stages of maturation were examined using both light (phase-contrast) and electron microscopy. At the germinal vesicle stage, CGs were distributed almost evenly in the cortex of the oocyte. A 'small' CG-free area of the cortex, with prominent cytoplasmic protrusions, appeared twice during the progression of meiosis. The first time, immediately above the metaphase spindle of the first meiosis and secondly, above the metaphase spindle of the second meiosis. Both peripheral migration and exocytosis of CGs appeared to be responsible for the formation of CG-free cortex above the metaphase spindle of first meiosis. The development of CG-free cortex above the metaphase spindle of the second meiosis was definitely due to exocytosis of CGs. The CG-free cortex above the metaphase spindle of the second meiosis increased its size steadily even after the oocytes had been transported into oviducts. Concomitantly, the size of the perivitelline space increased. The enlargement of the perivitelline space in unfertilized oviductal oocytes seems to be attributed not only to the exocytosis of CGs, but also to an extrusion of non-CG materials by the oocytes and perhaps an accumulation of some materials secreted by the oviduct epithelium. The limited (premature) CG-exocytosis during oocyte maturation and prior to fertilization may alter the physico-chemical properties of the zona pellucida slightly in such a way that the zona can be penetrated only by a very 'strong' spermatozoon. The presence of the perivitelline space in the mature oocyte prior to fertilization seems to be essential or at least beneficial to normal fertilization. Distinct cytoplasmic protrusions appear on the cortex above the metaphase spindle. The plasma membrane covering these protrusions seems to represent the membrane synthetized by the oocyte in preparation for the extrusion of polar body.

Animals↗
PubMed1985

Maturation of spermatozoa in the epididymis of the Chinese hamster.

Chinese hamster spermatozoa gain their ability to move when they descend from the testis to the distal part of the caput epididymis, but it is not until they enter the corpus epididymis that they become capable of fertilizing eggs. The maturation of the spermatozoa proceeds as they further descend the tract and perhaps continues even in the vas deferens. During transit between the distal caput and proximal cauda epididymides, small membrane-limited vesicles (and tubules) appear on the plasma membrane over the acrosomes of the spermatozoa. The number of vesicles appearing on the sperm brane reaches a maximum when the spermatozoa are in the proximal cauda epididymis. It declines sharply in the distal cauda epididymis. Spermatozoa in the vas deferens are free of the vesicles. The origin, chemical nature, and functional role of the vesicles that appear on the sperm surface during epididymal transit must be the subject of further investigation.

Animals↗
PubMed1985

Inner acrosomal membrane of mammalian spermatozoa: its properties and possible functions in fertilization.

The inner acrosomal membrane (IAM) develops during the spermatid stage of differentiation as that portion of the Golgi-derived acrosome granule that tightly associates with the condensing sperm nucleus. In some mammalian species, an electron-dense proteinaceous material accumulates between the IAM and the nuclear envelope, collectively comprising the "perforatorium." Evidence, including its partial purification and its structural resistance to detergents and sonication, suggests that the IAM is an unusually resiliant membrane. Dense paracrystalline arrays of intramembranous particles, a lack of lectin-mediated receptor modulation, and its lack of participation in sperm-egg fusion suggest that the IAM lacks the same degree of fluidity as the egg surface plasmalemma. Observations using monoclonal antibodies, however, suggest that some specific antigenic modulations may be possible within the IAM. Its structural rigidity is of obvious mechanical value during sperm penetration through the zone pellucida. An additional role as a scaffold for putative zona lysin material remains controversial. Biochemical evidence suggests that acrosin, for example, is not entirely soluble and that some remains sperm-associated, depending on the conditions of acrosome disruption. Nevertheless, morphological studies do not agree on acrosin's specific localization to the IAM. Currently there is only very limited information concerning the localization of the other acrosomal enzymes to the IAM. Another possible role for the IAM in some species may be in recognizing the zona pellucida. Evidence for this derives from the observation that fucoidin, a fucose heteropolysaccharide, inhibits guinea pig sperm-zona binding, and bound fucoidin can be localized to the IAM and equatorial regions of the living acrosome-reacted spermatozoa. Finally, the IAM may have a role in early recognition/adhesion with the colemma.

Acrosome↗
PubMed1985

pH and protease control of acrosomal content stasis and release during the guinea pig sperm acrosome reaction.

The purpose of this study was to examine how trypsin inhibitors affect the guinea pig sperm acrosome reaction in vitro. Using spermatozoa pretreated with lysophosphatidyl choline, we found that both naturally occurring high molecular weight and the smaller synthetic trypsin inhibitor p-aminobenzamidine (PAB) delayed the onset of the acrosome reaction as monitored by light microscopy. Examination with electron microscopy revealed that acrosomal matrix dispersal rather than membrane fusion was affected. Despite the morphologic delay in acrosomal content release, PAB unexpectedly permitted 96% of soluble acrosomal antigen to be released into the supernatant. In addition, total acrosin release in the presence of PAB was 74% of control, with the vast majority as latent rather than active enzyme. A morphologically intact but membrane-free target of acrosomal matrix (AM), which is sensitive to trypsin inhibitor, was partially purified using Triton-x-100 at pH 5.2. AM remained morphologically stable at pH 5.2; however, shift up to pH 7 resulted in rapid dissolution within several minutes as monitored by light and electron microscopy and light scattering. Trypsin inhibitor prevented dispersion of AM at pH 7. The results suggest that, during the acrosome reaction, one distinct region of the acrosomal contents disperses after membrane vesiculation in a pH and trypsin inhibitor-insensitive fashion while a pH sensitive trypsin-like activity (acrosin?) disperses another discrete region of acrosomal matrix.

Acrosin↗
PubMed1985

Fertility control in the bitch by active immunization with porcine zonae pellucidae: use of different adjuvants and patterns of estradiol and progesterone levels in estrous cycles.

To determine the changes in patterns of 17 beta-estradiol and progesterone levels underlying abnormal cycles in bitches immunized with solubilized crude porcine zonae pellucidae (cPZP), to attempt to circumvent these problems by immunizing with a purified zona fraction (pPZP), and to test the effectiveness of different adjuvants, bitches were immunized with cPZP or pPZP 2-6 times with no adjuvant, Freund's adjuvant, alum adjuvant, or the adjuvant CP-20,961. The bitch immunized without adjuvant had a low titer with a normal cycle and fertility. Immunization with cPZP and adjuvant produced moderate to high titers of antizona antibodies and infertility. Bitches with high titers experienced abnormal estrous cycles. Estradiol rose during proestrus, but instead of falling sharply in early estrus as in controls, it remained elevated. Progesterone did not rise. The moderate-titered bitches had normal cycles and steroid patterns. Bitches immunized with pPZP had moderate titers. Cycles were normal after 3 injections, but after 6 injections one bitch had an abnormal cycle. One pPZP-immunized bitch remained fertile but the others were infertile. Alum was the mildest adjuvant, causing no injection site lesions, but the highest titers occurred with Freund's and CP-20,961 adjuvants. All three adjuvants induced titers sufficient to inhibit fertility. Infertility in bitches immunized with PZP may be due to prevention of zona penetration, because their antisera inhibited zona penetration of oocytes by spermatozoa in vitro. However, alterations in ovarian function preventing ovulation and luteinization could be involved in high-titered bitches.

Adjuvants, Immunologic↗
PubMed1985

Multiple causes of pregnancy failure in hamsters precociously ovulated by human chorionic gonadotropin.

Cycling adult female hamsters can be induced to mate and ovulate 24 h early by the injection of 20 IU human chorionic gonadotropin (hCG) at 1500 h on Day 3 (day before proestrus), but pregnancy is not established. Although there is evidence of decreased sperm transport in precociously ovulated females, this does not appear to be the primary cause of infertility. Reduced size and vascularity of corpora lutea (CL) in treated females suggests incomplete or failed CL activation. Control and hCG-treated females were killed by exsanguination under ether anesthesia at intervals for the first 5 days after mating. Serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin, estradiol, and progesterone were measured by radioimmunoassay. Luteinizing hormone in treated animals was very high at 2200 h on Day 1 after mating (31 h after the hCG injection), due to endogenous release, and dropped below control levels thereafter. Follicle-stimulating hormone, by contrast, was significantly lower than controls at 2200 h on Day 1 and remained low until 2200 h on Day 3 after mating. Prolactin in treated animals was not different from that in controls, except for 1000 h on Day 4, when it showed a significant dip. Estradiol in treated animals was significantly higher than in controls at 2200 h on Day 1 (when LH was also high and FSH was low), and remained high at 1000 h and 2200 h on Day 2, dropping thereafter to control levels. Progesterone was initially at control levels but had dropped significantly by 1000 h on Day 2 and remained low for the next 24 h. These results suggest that pregnancy failure is due to inadequate activation of corpora lutea. This may be due to: 1) immaturity of follicles at the time of ovulation; 2) inappropriate timing of preovulatory events; 3) the luteolytic effects of high levels of LH or estradiol or both; 4) the low level of FSH in the early stages of corpus luteum development; or 5) a combination of the above. Abnormalities of prolactin secretion were not investigated in detail but cannot be ruled out at this time.

Animals↗
PubMed1984

Evidence suggesting the importance of fatty acids and the fatty acid moieties of sperm membrane phospholipids in the acrosome reaction of guinea pig spermatozoa.

When guinea pig spermatozoa were preincubated 1 hr in Ca2+-free medium containing dilysocardiolipin (100--125 micrograms/ml) then exposed to Ca2+, the majority underwent an immediate acrosome reaction. Monolysocardiolipin was much less effective and the native cardiolipin was totally ineffective. Some fatty acids added to the medium could also render the spermatozoa capable of undergoing their acrosome reactions, arachidonic acid in methyl ester form being most potent. It is known that sperm membrane contains phospholipase A which cleaves membrane phospholipids into lysophospholipids and fatty acids. Most lysophospholipids are known to be potent acrosome reaction-promoting agents. As some forms of fatty acids are also potent acrosome reaction-promoting agents, both products of membrane phospholipid hydrolysis by phospholipase A (i.e., both fatty acids and lysophospholipids) may work synergistically to effect the conversion of stable sperm membranes to a fusion competent state capable of engaging in the acrosome reaction.

Acrosome↗
PubMed1984

Fucoidin inhibits attachment of guinea pig spermatozoa to the zona pellucida through binding to the inner acrosomal membrane and equatorial domains.

Our previous study has shown that fucoidin, an algal heteropolysaccharide, is a potent inhibitor of sperm-zona binding in the guinea pig, hamster and human. To visualize the surface site of fucoidin binding, a biotinated derivative (B-Fuc) of the native fucoidin was prepared. B-Fuc retained the inhibitory activity and was used in conjunction with FITC-avidin to localize its binding sites on guinea pig spermatozoa using fluorescence microscopy. In living acrosome-reacted spermatozoa. B-Fuc bound predominantly to the inner acrosomal membrane and equatorial segment domains. The binding was effectively competed by a 10-fold excess of native fucoidin, but not by a 10-fold excess of heparin or a 20-fold excess of biotinated normal rabbit serum IgG. B-Fuc binding patterns on dead spermatozoa were quite different from that of living spermatozoa. The post-acrosomal region, rather than the inner acrosomal membrane and equatorial domains, was intensely labeled. This indicates the importance of using living cells in assessing true surface binding sites whenever possible. We conclude that the inner acrosomal membrane and/or equatorial domains are critical for zona binding in the guinea pig.

Acrosome↗
PubMed1984

Localization of thiol and disulfide groups in guinea pig spermatozoa during maturation and capacitation using bimane fluorescent labels.

The distribution of thiols and disulfides in the guinea pig spermatozoon during maturation and capacitation was studied using both membrane-permeable (mBBr) and impermeable (qBBr) forms of bromobimane, a specific fluorescent probe for thiol groups. In conjunction with the disulfide (SS)-reducing agent dithiothreitol (DTT) and the thiol-alkylating agent N-ethylmaleimide (NEM), quantitative spectrofluorometric measurements of the relative amounts of total thiol (SH) versus SS were performed on cauda epididymal spermatozoa. Under conditions labeling 70% of the reactive thiols, the ratio total SS/SH was 2.4/1.0. Contamination by other cell types prevented similar measurements on spermatozoa at earlier stages of epididymal maturation; thus, the qualitative localization of SH and SS groups in these and in capacitated spermatozoa was visualized using fluorescence microscopy. As spermatozoa moved from the testis to the caput epididymidis, there was a slight apparent increase in staining both on the surface and internally in all regions. Thereafter, surface and internal staining decreased by the time spermatozoa reached the cauda epididymidis. Fluorescence patterns were unaltered under short-term (1 h) capacitation conditions in calcium-free modified Tyrode's medium containing lysophosphatidyl choline and after induction of the acrosome reaction with 2 mM calcium. However, long-term capacitation (16-18 h) in calcium-free modified Tyrode's medium resulted in a loss of detectable SH in the head and acrosome. Regardless of the stage examined, sperm tails contained the greatest relative amount of SH, followed by the head and the acrosome. In addition, there was always more SH detectable internally than on the surface. DTT pretreatment caused a dramatic increase in staining in all regions, both surface and internal, consistent with the quantitative estimates of the SS/SH ratio.

Animals↗
PubMed1983

Freeze-fracture observations of ovulated hamster oocytes with their cumulus cells.

Mature unfertilized oocytes in the cumulus oophorus were examined using the freeze-fracture technique. The P face of the oocyte plasma membrane (oolemma) has randomly distributed intramembranous particles (IMPs) with an average diameter of about 10 nm. No marked difference is found between the intermicrovillous and microvillous regions of the oocyte with respect to the size and distribution of IMPs. The E face of the oolemma has fewer IMPs than the P face. The intermicrovillous region is slightly elevated in some places because of close contact with underlying cortical granules. The oolemma of this elevated region and the membrane of the cortical granule have IMPs which are considerably smaller than those in the remaining areas of the oolemma. The IMP density of the P face of the cortical granule membrane is much lower than that of the E face, possibly indicating a membrane specialization required for fusion of the cortical granule membrane with the overlying oolemma at the time of exocytosis. Cytoplasmic lamellae appear as parallel bands of particles in freeze-fractured specimens. No gap junctions are found between the oocyte and cumulus cells, but residual gap junctions are seen among clustered cumulus cells.

Animals↗
PubMed1983

What functions of the sperm cell are measured by in vitro fertilization of zona-free hamster eggs?

Human spermatozoa were capacitated in media containing either high concentrations (3.5%) of human serum albumin (HSA) or low concentrations (0.3%) of bovine serum albumin (BSA). The effects of both capacitation media were assessed immediately and after overnight preincubation (18 to 24 hours) by adding a mixture of nonliving human oocytes and living zona-free hamster eggs to the sperm suspension. Overnight preincubation of sperm in media containing 3.5% HSA enhanced sperm fusion with hamster vitelli, but the capacity for zona penetration was lost. These effects could be attributed to the concentration of HSA rather than a general effect of albumin concentration, because overnight preincubation in 3.5% BSA did not interfere with zona penetration. During overnight incubation in 3.5% HSA, the percentage of acrosome reactions increased, as did alterations in the equatorial segment of the acrosome-reacted sperm. The percentage of motile sperm remained high after overnight incubation in 3.5% HSA, but both the mean swimming speed and flagellar activity on the zona surface declined. The sperm also lost their ability for strong zona binding after overnight incubation in 3.5% HSA.

Animals↗
PubMed1982

Infertility in bitches induced by active immunization with porcine zonae pellucidae.

In a study designed to evaluate the contraceptive potential of anti-egg zona pellucida immunization, bitches were injected with isolated and solubilized zonae pellucidae of either the pig or the dog in saline and Freund's adjuvant or with saline and adjuvant alone (controls). They were boosted monthly, and serum samples were collected before the first injection and 10 days after each injection. The titers of anti-zona pellucida antibodies in each serum sample were measured by treating fresh canine oocytes with the serum, then evaluating antibody binding as indicated by indirect immunofluorescence, precipitation of the zona surface, and penetrability of the zonae by spermatozoa in vitro. The bitches were bred when they came into estrus. All three bitches immunized with porcine zonae developed high titers (1:10,000 or more by indirect immunofluorescence) of antibodies that cross-reacted with canine zonae to cause precipitation of the zona surface both in vivo and in vitro and that completely inhibited penetration of the zonae by spermatozoa in vitro. The two bitches immunized with canine zonae developed only low titers, and their sera had little or no effect on treated zonae. The two control bitches did not develop anti-zona antibody. None of the bitches immunized against porcine zonae became pregnant when bred, but one bitch immunized against canine zonae and one control did become pregnant. The bitches immunized with porcine zonae had somewhat abnormal cycles for unknown reasons. Thus, we could not establish with certainty whether the infertility resulted from specific interference with fertilization, as in vitro, or from alterations in ovarian function, or both.

Animals↗
PubMed1982

Fertile life of acrosome-reacted guinea pig spermatozoa.

To study the fertile life of acrosome-reacted guinea pig spermatozoa, spermatozoa were induced to undergo a synchronous acrosome reaction after which vigorously motile acrosome-reacted spermatozoa were isolated from acrosome-intact ones. Acrosome-reacted spermatozoa thus prepared were incubated in vitro for various periods of time before they were mixed with zona-intact and zona-free eggs. Such spermatozoa retained their ability to fertilize zona-intact eggs for at least 2 hours after the acrosome reaction; thereafter they rapidly lost this ability. There was a close correlation between the decline and loss of the ability to penetrate the zona and the loss of hyperactivated motility by spermatozoa, indicating the importance of vigorous motility in passing through the zona. The ability of acrosome-reacted spermatozoa to fertilize zona-free eggs was maintained for many hours even after the spermatozoa had completely lost their capacity to fertilize zona-intact eggs. As long as acrosome-reacted spermatozoa are "alive," they seem to retain the capacity to fuse with the egg plasma membrane regardless of the quality of their motility.

Acrosome↗
PubMed1982

Sperm autoantigens and fertilization III. Ultrastructural localization of guinea pig autoantigens.

Guinea pig sperm autoantigens have been localized by direct and indirect immunoferritin techniques in (1) plasma membrane over the entire sperm head, (2) acrosomal contents, (3) fibrous sheath and outer dense fibers of the tail filament, and (4) the inner acrosomal membrane of 50% of acrosome reacted spermatozoa. These cellular structures are known to be involved in guinea pig sperm rouleaux formation, acrosome reaction, interaction of acrosome-reacted sperm with zona pellucida and with the vitellus of guinea pig ova. Since IgG and Fab of autoantiserum to guinea pig spermatozoa have been shown to interfere with these cellular reactions, this study provides further evidence, albeit indirect, that sperm autoantigens are involved in these cellular events.

Acrosome↗
PubMed1982

[Effects of agents used for removal of zona pellucida on human sperm penetration into zona-free hamster egg].

In 1976, Yanagimachi et al. suggested that human sperm were capable of penetrating zona-free hamster eggs. Since that time, this in vitro penetration assay has been used to analyze human spermatozoal fertilizing ability. In the present study, effects of proteinases and S-S reducing agents used for removal of zona pellucida on human sperm penetration into zona-free hamster eggs were investigated. 0.1% trypsin, 0.05% pronase, 0.1% alpha-chymotrypsin and 300 IU/ml Dispase were proteinases used for this experiment, and 0.05 M mercaptoethanol and 0.025 M dithiothreitol (DDT) were S-S reducing agents. Results obtained were as follows: 1) When hamster eggs were treated for 3 minutes by these agents, percentages of eggs penetrated were 76.4% (trypsin), 75.6% (mercaptoethanol), 64.7% (alpha-chymotrypsin), 63% (pronase), 61% (Dispase) and 25.9% (DTT). 2) When hamster eggs were treated for 20 minutes, the figures were 83% (mercaptoethanol), 75.7% (trypsin), 63% (Dispase) 54% (pronase), 51% (alpha-chymotrypsin) and 13% (DTT). 3) There was no difference between human sperm penetration rates into zona-free hamster eggs treated for 3 minutes and those treated for 20 minutes. These results suggest that trypsin and mercaptoethanol were very useful agents for removing zona pellucida of hamster eggs in this in vitro fertilization system.

Animals↗